Invasive Fungal Infections (IFI) are an important cause of disease, especially in immuncompromised patients, including patients undergoing high dose chemotherapy, patients receiving stem cell and bone marrow transplants, preterm neonates, intensive care patients, and patients with acquired or innate immune deficiencies. Fungal, bacterial, viral, helminth, and other infections each require different courses of treatment and choosing the wrong treatment could cause unnecessary side effects and extend patient suffering. Clearly, a clinician needs a rapid, single diagnostic test to differentiate fungal infections from infections with other microorganisms.
Using current techniques, fungal infections are difficult to diagnose in clinical setting because fungi are difficult to culture, with the time of culture often extending beyond clinical utility and with culture failures (rendering no useful diagnostic information) frequent. (Preuner and Lion, Expert Rev. Mol. Diagn. 9, 397-401, 2009). This results in an under-diagnosis and under-treatment of fungal infections.
PCR amplification techniques have been used to detect fungal nucleic acids directly isolated from samples without the need for culture. The development of these techniques has been hindered by fungal contaminants that inhibit PCR (Borman et al, J Antimicrob Chemother 61 17-112, 2008) or by appropriate experimental controls that would allow the researcher to detect the presence of these or other contaminants (Khot and Fredricks Expert Rev Anti Infect Ther 7, 1201-1221, 2009).
A further challenge to the development of PCR assays for fungal infection is the development of a single, broad range PCR assay that detects a wide variety of fungi including a wide range of infective fungi strains and species, fungi not normally known to infect human beings, or even fungi that are not characterized. Such an assay must be broad enough to amplify the vast majority of known fungal species (including those that are difficult to culture,) but selective enough that contaminating human DNA or other contaminating DNA are not significantly amplified. Many attempts have been made, but none have done so using degenerate PCR primers. Additionally, such an assay must also be able to quantify the fungal load to provide additional clinical utility and the ability to create a clonal library for identification and quantification of individual fungal species.